Cis-2-dodecenoic Acid Mediates Its Synergistic Effect with Triazoles by Interfering with Efflux Pumps in Fluconazole-resistant Candida albicans / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the synergy of the Burkholderia signaling molecule cis-2-dodecenoic acid (BDSF) and fluconazole (FLU) or itraconazole (ITRA) against two azole-resistant C. albicans clinical isolates in vitro and in vivo.@*METHODS@#Minimum inhibitory concentrations (MICs) of antibiotics against two azole-resistant C. albicans were measured by the checkerboard technique, E-test, and time-kill assay. In vivo antifungal synergy testing was performed on mice. Analysis of the relative gene expression levels of the strains was conducted by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).@*RESULTS@#BDSF showed highly synergistic effects in combination with FLU or ITRA with a fractional inhibitory concentration index of ⪕ 0.08. BDSF was not cytotoxic to normal human foreskin fibroblast cells at concentrations of up to 300 μg/mL. The qRT-PCR results showed that the combination of BDSF and FLU/ITRA significantly inhibits the expression of the efflux pump genes CDR1 and MDR1 via suppression of the transcription factors TAC1 and MRR1, respectively, when compared with FLU or ITRA alone. No dramatic difference in the mRNA expression levels of ERG1, ERG11, and UPC2 was found, which indicates that the drug combinations do not significantly interfere with UPC2-mediated ergosterol levels. In vivo experiments revealed that combination therapy can be an effective therapeutic approach to treat candidiasis.@*CONCLUSION@#The synergistic effects of BDSF and azoles may be useful as an alternative approach to control azole-resistant Candida infections.

Protective Effects of cis-2-Dodecenoic Acid in an Experimental Mouse Model of Vaginal Candidiasis / 生物医学与环境科学（英文）

OBJECTIVE@#To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo.@*METHODS@#The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 μmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms.@*RESULTS@#In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 μmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 μmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 μmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively.@*CONCLUSION@#BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.

Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I / 华中科技大学学报（医学）（英德文版）

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

Pentoxifylline inhibits liver fibrosis via hedgehog signaling pathway / 华中科技大学学报（医学）（英德文版）

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

Involvement of TLR2-MyD88 in abnormal expression of miR-146a in peripheral blood monocytes of patients with chronic hepatitis C / 华中科技大学学报（医学）（英德文版）

miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.

Involvement of TLR2-MyD88 in abnormal expression of miR-146a in peripheral blood monocytes of patients with chronic hepatitis C / 华中科技大学学报（医学）（英德文版）

miR-146a is an immunoregulatory microRNA closely associated with viral infection. This study investigated the expression changes of miR-146a in peripheral blood monocytes of HCV-infected patients and the mechanism by which the THP-1 cells were stimulated with HCV core protein in vitro. It was found that in the peripheral blood monocytes of HCV-infected patients, miR-146a expression was upregulated. After treated by interferon/ribavirin, miR-146a expression was decreased when HCV RNA became undetectable. HCV core could directly stimulate THP-1 cells to produce miR-146a. Silencing TLR2 and MyD88 could significantly inhibit the expression of miR-146a. It was concluded that the expression of miR-146a in peripheral blood monocytes of HCV-infected patients was abnormally increased. The TLR2-MyD88 signaling pathway may take part in the overexpression of miR-146a in monocytes stimulated with HCV core protein.

Upregulation of toll-like receptor 4 on T cells in PBMCs is associated with disease aggravation of HBV-related acute-on-chronic liver failure / 华中科技大学学报（医学）（英德文版）

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4(+) and CD8(+) T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4(+) and CD8(+) T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4(+) T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse / 华中科技大学学报（医学）（英德文版）

The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.

Chronic HBV carrier's acceptance of regular surveilling program in China / 华中科技大学学报（医学）（英德文版）

Long-term compliance with regular surveillance is important for the prevention and timely management of chronic hepatitis B (CHB). However, there are no researches focusing on the compliance of hepatitis B virus infected patients in regular surveillance so far. The purpose of our study was to investigate the outpatient compliance with long-term regular surveillance in China. Data of 3257 CHB outpatients was pooled and analyzed to assess the outpatient's compliance with the long-term regular surveillance plan. In all outpatients, the non-follow-up and the follow-up group accounted for 73.2% and 26.8%, respectively. Among the follow-up outpatient's, only 48.9% received ongoing-follow-up and 51.1% were finally lost to follow-up; the median length of visiting duration was 25 months; and the predictive 1-, 2-, 3-, 4- and 5-year ongoing follow-up rate was 72.7%, 52.5%, 42.4%, 33.8%, and 26.3%, respectively. In conclusion, our survey proved that the regular long-term surveillance on Chinese chronic HBV carrier is difficult to be fully implemented. A large proportion of outpatients do not receive routine follow-up and are at risk of treatment delay due to various social reasons.

LY294002 enhances inhibitory effect of gemcitabine on proliferation of human pancreatic carcinoma PANC-1 cells / 华中科技大学学报（医学）（英德文版）

Phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely applied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 cells was investigated. The expression of PI3K, phosphorylated Akt (p-Akt) and multidrug-resistance like protein (MRP) in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was detected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and protein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pancreas tissues and chronic pancreatitis tissues (P<0.01 and P<0.05 respectively). LY294002 could effectively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.

LY294002 enhances inhibitory effect of gemcitabine on proliferation of human pancreatic carcinoma PANC-1 cells / 华中科技大学学报（医学）（英德文版）

Phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB, Akt) pathway plays a major role in proliferation and survival of many types of cells. The inhibitory effect of LY294002, widely applied as an inhibitor of PI3K, in combination with gemcitabine on proliferation of PANC-1 cells was investigated. The expression of PI3K, phosphorylated Akt (p-Akt) and multidrug-resistance like protein (MRP) in normal pancreas tissues, chronic pancreatitis tissues and pancreatic carcinoma tissues was detected. The effects of LY294002 combined with gemcitabine on proliferation of PANC-1 cells and protein levels of p-Akt and MRP were detected. The results showed that the positive expression rate of PI3K, p-Akt and MRP in pancreatic carcinoma tissues was significantly higher than that in normal pancreas tissues and chronic pancreatitis tissues (P<0.01 and P<0.05 respectively). LY294002 could effectively enhance the inhibitory effect of gemcitabine on proliferation of PANC-1 cells. Furthermore, Western blotting revealed that LY294002 combined with gemcitabine reduced the protein levels of p-Akt and MRP, which contributed to the inhibition of proliferation. It is concluded that LY294002 in combination with gemcitabine may represent an alternative therapy for pancreatic carcinoma.

Chronic HBV carrier's acceptance of regular surveilling program in China / 华中科技大学学报（医学）（英德文版）

Long-term compliance with regular surveillance is important for the prevention and timely management of chronic hepatitis B (CHB). However, there are no researches focusing on the compliance of hepatitis B virus infected patients in regular surveillance so far. The purpose of our study was to investigate the outpatient compliance with long-term regular surveillance in China. Data of 3257 CHB outpatients was pooled and analyzed to assess the outpatient's compliance with the long-term regular surveillance plan. In all outpatients, the non-follow-up and the follow-up group accounted for 73.2% and 26.8%, respectively. Among the follow-up outpatient's, only 48.9% received ongoing-follow-up and 51.1% were finally lost to follow-up; the median length of visiting duration was 25 months; and the predictive 1-, 2-, 3-, 4- and 5-year ongoing follow-up rate was 72.7%, 52.5%, 42.4%, 33.8%, and 26.3%, respectively. In conclusion, our survey proved that the regular long-term surveillance on Chinese chronic HBV carrier is difficult to be fully implemented. A large proportion of outpatients do not receive routine follow-up and are at risk of treatment delay due to various social reasons.

Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse / 华中科技大学学报（医学）（英德文版）

The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.

Assessment of mitochondrial toxicity induced by zidovudine and adefovir dipivoxil in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mitochondrial toxicities induced by zidovudine (AZT) and adefovir dipivoxil (ADV) antiviral drugs using a rat model system.</p><p><b>METHODS</b>Twelve healthy Sprague-Dawley rats were randomly divided into three equal groups and treated by oral gavage with zidovudine (125 mg/kg/day), adefovir (40 mg/kg/day), or saline (equal volume) for 28 days. The rats' body weights were measured once a week, and blood was collected every two weeks for blood and biochemical tests. All animals were sacrificed at the end of treatment, and liver, kidney, skeletal muscle, and cardiac muscle were collected by necropsy. Mitochondria were isolated from the respective tissue samples, and the activities of respiratory chain complexes were measured. DNA was purified from each sample and the mitochondrial DNA (mtDNA) content was monitored by quantitative real time PCR. Mitochondrial morphology was analyzed under electron microscope.</p><p><b>RESULTS</b>No significant adverse effects, including body weight loss, abnormal blood or biochemistry, were observed in rats treated with AZT or ADV. The activities of mitochondrial cytochrome c oxidase in liver and cardiac muscle were slightly decreased in rats treated with AZT (liver: 9.44+/-3.09 vs. 17.8+/-12.38, P?=?0.21; cardiac muscle: 32.74+/-5.52 vs. 24.74+/-20.59, P?=?0.28; kidney: 4.42+/-1.53 vs. 14.45+/-13.75, P?=?0.18; skeletal muscle: 33.75+/-8.74 vs. 40.04+/-2.49, P?=?0.45). The mtDNA content was significantly decreased in cardiac muscle of AZT-treated rats (cardiac muscle: 0.15+/-0.13 vs. 0.32+/-0.42, P?=?0.85). The morphology of mitochondria in liver, kidney, skeletal muscle, and cardiac muscle was significantly altered in the AZT-treated rats and included disappearance of the outer membrane, severely damaged structure, and swollen or completely absent cristae. No obvious effects were noted in the ADV- or saline-treated rats.</p><p><b>CONCLUSION</b>Significant adverse effects related to mitochondrial toxicity were observed in rats treated with AZT. The slightly decreased mtDNA content in ADV-treated rats may suggest that this antiviral drug can also cause mitochondrial toxic effects.</p>

The influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and antiviral proteins of IFN alpha / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha.</p><p><b>METHODS</b>The HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment.</p><p><b>RESULTS</b>The HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells.</p><p><b>CONCLUSIONS</b>The HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.</p>

Characterization of HBsAbs in occult hepatitis B virus infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the properties of HBsAb in occult hepatitis B virus infection and its affinity to different serotypes of hepatitis B virus surface antigen (HBsAg).</p><p><b>METHODS</b>Long-term follow-up was conducted in 2 HBsAb positive patients with occult hepatitis B virus infection. HBsAg was detected using multiple diagnostic kits and the HBsAb subtype was determined by performing neutralization experiments with different serotypes of HBsAg. The viral S gene was PCR-amplified and mutation analysis was conducted. Plasmids expressing HBsAgs were constructed by inserting these PCR products into an eukaryotic expression vector and were then transfected into HepG2 cells. The cell culture supernatant and cellular extracts were detected for HBsAg respectively. Neutralization experiments were carried out in the cell culture supernatant from HBsAg plasmids transfected HepG2 cells and serum samples from these patients and others who had been confirmed to be positive for HBsAb.</p><p><b>RESULTS</b>Multiple tests using various diagnostic kits showed that the 2 patients were negative for HBsAg and the three different serotypes of HBsAg (adr, adw, ay) could neutralize 82.1%-100% of HBsAb existed in the 2 patients. Sequence analysis of S gene cloned from these patients revealed that the homology to reference strain were 95.13%-97.79% and 92.04%-95.58% respectively at the nucleotide and amino acid levels. Quantitation of HBsAg showed that the expression levels of HBsAg from the two patients were 41.1% and 22.6% respectively of that of control HBsAg in cell culture supernatant and 48.1% and 59.3% respectively in cellular extract, and the supernatant/cell lysate ratios were 0.85 and 0.38 respectively. In neutralization experiments, HBsAg could be totally absorbed by control serum, whereas could only be partially neutralized by HBsAbs from the two patients (F = 353.6 and 645.2, P is less than 0.01).</p><p><b>CONCLUSION</b>Both the antigenicity and the ability of HBsAg secreted outside of the cells are decreased in these HBsAb-positive patients with occult HBV infection. The HBsAbs are mainly specific for common epitopes among different serotypes of HBsAg and are probably different as compared with those produced by vaccine inoculation.</p>

Based on the low-density cDNA Macroarray for screening of antiviral proteins of IFNa tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To screen the gene expression profiles of IFN-alpha antiviral proteins based on a low-density cDNA Macroarray, and to explore the relationship between the expression of antiviral protein and the HBV replication.</p><p><b>METHODS</b>The HepG2 and HepG2.2.15 cells were treated with various concentrations of IFN-alpha (0 IU/ml, 100 IU/ml, 1000 IU/ml) of IFN-alpha for 6 h, and then the low-density cDNA Macroarray was used for analysing the expression profiles of antiviral genes and screening differential expressions of antiviral proteins. Meanwhile, the HepG2 cells were transiently transfected with HBV core protein-expressed plasmid pHBc-EGFP, and the expressions of antiviral proteins were analysed by RT-PCR assay. Moreover, the HepG2.2.15 cells were also transfected with the antiviral protein-expressed plasmid pcDNA3.1-Flag-MxA. ELISA was used for analysing the secreted HBV antigens, while dot blot and Southern blot were applied for analysing the extracellular HBV DNA and intracellular replicative intermediate HBV DNA in HepG2.2.15 cells. All data were presented as mean+/-SD and analyzed using the t-test and one-way analysis of variance (ANOVA) in the experiments.</p><p><b>RESULTS</b>The Macroarray results suggested that the expression of IFN-alpha antiviral genes like 6-16, IFITM1, IFITM2, IFITM3 and RING4 in HepG2.2.15 cells were partially inhibited. More importantly, it was found, in this research, the expression of antiviral protein MxA in HepG2.2.15 cells was completely suppressed. RT-PCR analysis indicated that the expression of MxA was also significantly decreased in HepG2 cells transfected with pHBc-EGFP plasmid. Although HepG2.2.15 cells transfected with pcDNA3.1-Flag-MxA plasmid could not inhibit extracellular HBV DNA and intracellular replicative intermediate HBV DNA, the MxA exerted some antiviral activities as it effectively suppressed the secretion of HBsAg and HBeAg in HepG2.2.15 cells.</p><p><b>CONCLUSIONS</b>HBV and its antigen components probably influence the expression of antiviral proteins. IFN- resistance may be related to the down-regulation of antiviral proteins expression.</p>

Toll-like receptor dependent innate immune responses by primary mouse hepatocytes and its control of HBV replication / 中华肝脏病杂志

<p><b>OBJECTIVE</b>This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.</p><p><b>METHODS</b>We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).</p><p><b>RESULTS</b>We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.</p><p><b>CONCLUSION</b>Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.</p>

S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients.</p><p><b>METHODS</b>HBV S gene from 8 patients (Group A) with HBsAg (+)/HBsAb (+) and 9 patients (Group B) with HBsAg (+)/HBsAb (-)was amplified by polymerase chain reaction (PCR) and sequencing. Both the distribution of genotype and serotype and the rate of MHR region were compared by Fisher's exact test. The mutation rate of both the DNA level and amino acid level was compared by t test.</p><p><b>RESULTS</b>No significant difference in distribution of genotypes was found between the two groups (P=0.153). In group A, 2 were genotype B, 6 were genotype C; In group B, 6 were genotype B, 3 were genotype C. No significant difference in distribution of serotypes was found between the two groups, either (P=0.218). In group A, 2 were adw, 5 were adr, 1 was ayr; In group B, 6 were adw, 3 were adr. The mutation rate of Pre-S1 region at both the DNA level (2.29% vs 1.80%, t=2.66, P more than 0.05) and the amino acid level (2.66% vs 1.59%, t=1.39, P>0.05) was not significantly different between these two groups; the mutation rate of Pre-S2 region in group A patients was significantly higher than that in group B at the DNA level (1.74% vs 0.91%, t=4.68, P<0.01), but not higher at the amino acid level (3.18% vs 2.05%, t=1.85, P>0.05), the mutation rate of S region in group A patients was significantly higher than that in group B at both the DNA level (2.13% vs 0.81%, t=6.00, P<0.01) and the amino acid level (4.37% vs 1.52%, t=5.32, P<0.01). Amino acid substitutions were found both within and beyond the MHR region. The rate of "a" determinant mutations in these two groups was also found to be significantly different (P<0.05).</p><p><b>CONCLUSION</b>Higher HBV S gene mutation rate exists in HBsAg/HBsAb double positive patients than that in HBsAg (+)/HBsAb (-) patients.</p>

The immunization effects of HBV core antigen and surface antigen fusion protein in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein.</p><p><b>METHODS</b>The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined.</p><p><b>RESULTS</b>The plasmid containing the chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also caused significant HBcAg specific lymphocyte proliferation. Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg can induce more effective cellular response but weaker humoral response.</p><p><b>CONCLUSION</b>Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.</p>